Method of assisting in diagnosis of nonalcoholic steatohepatitis and method of obtaining data for assisting in diagnosis of nonalcoholic steatohepatitis

Abstract

To provide a method of assisting in diagnosis of nonalcoholic steatohepatitis by measuring the levels of specific markers in a sample, and a method of obtaining data for assisting in diagnosis of nonalcoholic steatohepatitis by measuring the levels of specific markers in the sample.SOLUTION: A method of assisting in diagnosis of nonalcoholic steatohepatitis is provided, the method comprising a step 1 of measuring the level of LDL in a sample and the level of phosphatidylcholine in the LDL in the sample, and a step 2 of deriving a ratio of the LDL level and the phosphatidylcholine level in the LDL.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for aiding in the assessment of non-alcoholic steatohepatitis and to a method for obtaining data for aiding in the assessment of non-alcoholic steatohepatitis. [Background technology]

[0002] Nonalcoholic fatty liver disease (NAFLD) is a disease in which fatty liver is detected by histological and imaging diagnosis. In recent years, the number of patients in Japan has been increasing due to changes in lifestyle. NAFLD is divided into simple fatty liver (NAFL), in which the disease progresses very little, and nonalcoholic steatohepatitis (NASH), which progresses to cirrhosis and liver cancer. Diagnosis of NASH requires histopathological diagnosis by liver biopsy, but liver biopsy is highly invasive and places a heavy burden on patients. Methods for diagnosing fatty liver disease by analyzing blood-derived samples have been reported.

[0003] Patent document 1 describes a method for diagnosing fatty liver disease, which includes the steps of analyzing at least one selected from the group consisting of chylomicrons, very low density lipoproteins, chylomicron remnants, and very low density lipoprotein remnants contained in a blood-derived sample collected from a subject, and determining the incidence of fatty liver disease, the severity of the disease, or the effectiveness of treatment for the disease based on the results of the analysis.

[0004] Patent Document 2 describes a method for diagnosing non-alcoholic steatohepatitis, which is characterized by measuring the phospholipids or fatty acid composition of phospholipids in peripheral blood.

[0005] Patent Document 3 describes a method for providing information regarding the histological diagnosis of severity or prognosis of non-alcoholic fatty liver disease (NAFLD), which includes a measurement step of measuring the content of sphingomyelin (SM) in a biological sample separated from a specimen.

[0006] Furthermore, Patent Document 4 describes a method for assisting in the detection of non-alcoholic steatohepatitis, which comprises measuring the amount of LDL-TG (triglycerides in low-density lipoproteins) and / or ApoE-rich HDL-C (cholesterol in high-density lipoproteins rich in apolipoprotein E) present in a test blood sample isolated from a living body. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. WO2009 / 048081 [Patent Document 2] International Publication No. WO2005 / 109006 [Patent Document 3] Special Publication No. 2022-502685 [Patent Document 4] Patent No. 7029139 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for assisting in the diagnosis of non-alcoholic steatohepatitis by measuring the amount of a predetermined marker in a sample.A further object of the present invention is to provide a method for obtaining data for assisting in the diagnosis of non-alcoholic steatohepatitis by measuring the amount of a predetermined marker in a sample. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the present inventors have found that the ratio of the amount of LDL in a sample to the amount of phosphatidylcholine in the LDL in the sample can be used as an index to assist in the diagnosis of non-alcoholic steatohepatitis. The present invention was completed based on the above findings.

[0010] According to the present invention, the following inventions are provided. <1> A step 1 of measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample; Step 2: calculating the ratio between the amount of LDL and the amount of phosphatidylcholine in LDL A method for assisting in the determination of non-alcoholic steatohepatitis, comprising: <2> If the ratio of LDL to phosphatidylcholine in LDL is equal to or greater than the cutoff value, it indicates a high possibility of developing nonalcoholic steatohepatitis. <1> The method described below. <3> The ratio of the amount of LDL to the amount of phosphatidylcholine in the LDL is used as an index, and if the ratio is equal to or greater than the ratio of the amount of LDL in a sample from a patient suffering from simple fatty liver to the amount of phosphatidylcholine in the LDL in the sample, it indicates a high possibility that the patient has developed non-alcoholic steatohepatitis. <1> The method described below. <4> Phosphatidylcholine in LDL total phosphatidylcholine, or one or more types of phosphatidylcholine in which the total number of carbon atoms in the hydrocarbon groups constituting the fatty acids in the phosphatidylcholine is 30 to 40 and the total number of double bonds in the hydrocarbon groups constituting the fatty acids is 0 to 7; <1> The method described below. <5> To help distinguish non-alcoholic steatohepatitis from simple fatty liver disease; <1> The method described below. <6> The specimen is a blood sample; <1> The method described below. <7> The specimen is whole blood, serum, or plasma; <1> The method described below. <8> A step 1 of measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample; Step 2: calculating the ratio between the amount of LDL and the amount of phosphatidylcholine in LDL A method for obtaining data to assist in the diagnosis of non-alcoholic steatohepatitis, comprising: <9> Phosphatidylcholine in LDL total phosphatidylcholine, or one or more types of phosphatidylcholine in which the total number of carbon atoms in the hydrocarbon groups constituting the fatty acids in the phosphatidylcholine is 30 to 40 and the total number of double bonds in the hydrocarbon groups constituting the fatty acids is 0 to 7; <8> The method described below. <10> The specimen is a blood sample; <8> The method described below. <11> The specimen is whole blood, serum, or plasma; <8> The method described below. [Effects of the Invention]

[0011] According to the present invention, it is possible to assist in the diagnosis of non-alcoholic steatohepatitis with high sensitivity and high specificity. According to the present invention, it is possible to obtain data for assisting in the diagnosis of non-alcoholic steatohepatitis with high sensitivity and high specificity. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of measurement by liquid chromatography mass spectrometry in Example 1. [Figure 2] FIG. 2 shows the results of the ROC analysis in Example 1. [Figure 3] FIG. 3 shows the measurement results of liquid chromatography mass spectrometry in Example 2. [Figure 4] FIG. 4 shows the measurement results of liquid chromatography mass spectrometry in Example 2. [Figure 5] FIG. 5 shows the measurement results of liquid chromatography mass spectrometry in Example 2. [Figure 6] FIG. 6 shows the measurement results of liquid chromatography mass spectrometry in Example 2. [Figure 7] FIG. 7 shows the results of the ROC analysis in Example 2. [Figure 8] FIG. 8 shows the results of the ROC analysis in Example 2. [Figure 9] FIG. 9 shows the results of the ROC analysis in Example 2. [Figure 10]FIG. 10 shows the results of the ROC analysis in Example 2. [Figure 11] FIG. 11 shows the measurement results of liquid chromatography mass spectrometry in Example 3. [Figure 12] FIG. 12 shows the measurement results of liquid chromatography mass spectrometry in Example 3. [Figure 13] FIG. 13 shows the results of the ROC analysis in Example 3. [Figure 14] FIG. 14 shows the results of the ROC analysis in Example 3. [Figure 15] FIG. 15 shows the measurement results of liquid chromatography mass spectrometry in Example 4. [Figure 16] FIG. 16 shows the measurement results of liquid chromatography mass spectrometry in Example 4. [Figure 17] FIG. 17 shows the results of the ROC analysis in Example 4. [Figure 18] FIG. 18 shows the results of the ROC analysis in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. In this specification, the symbol "to" indicates a range that includes the numerical values ​​before and after it as the minimum and maximum values, respectively. As used herein, LDL means Low Density Lipoprotein.

[0014] As used herein, phosphatidylcholine refers to a compound represented by the following formula (1):

[0015] [ka]

[0016] In formula (1), R1 and R2 each independently represent a hydrocarbon group constituting a fatty acid. The total number of carbon atoms in the hydrocarbon groups constituting the fatty acid represented by R1 and R2 is generally 2 to 50, preferably 20 to 45, and more preferably 30 to 40. The fatty acid may be either a saturated fatty acid or an unsaturated fatty acid. The hydrocarbon group constituting the fatty acid may be linear, branched, cyclic, or a combination thereof. The number of carbon atoms in R1 and the number of carbon atoms in R2 may be the same or different. In the present invention, the "hydrocarbon group constituting the fatty acid in the phosphatidylcholine" refers to the R 1 and R 2 is equivalent to

[0017] The molecular species 30:0 of ​​phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 28, and the total number of unsaturated bonds in R1 and R2 is 0. An example of the molecular species 30:0 is a molecule of formula (1) in which R1 has 14 carbon atoms, R2 has 14 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 0.

[0018] The molecular species 32:0 of ​​phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 30, and the total number of unsaturated bonds in R1 and R2 is 0. An example of the molecular species 32:0 is a molecule of formula (1) in which R1 has 15 carbon atoms, R2 has 15 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 0. The molecular species 32:1 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 30, and the total number of unsaturated bonds in R1 and R2 is 1. An example of the molecular species 32:0 is a molecule of formula (1) in which R1 has 15 carbon atoms, R2 has 15 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 1. The molecular species 32:2 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 30, and the total number of unsaturated bonds in R1 and R2 is 2. An example of the molecular species 32:2 is a molecule of formula (1) in which R1 has 15 carbon atoms, R2 has 15 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 2.

[0019] The molecular species 34:1 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 32, and the total number of unsaturated bonds in R1 and R2 is 1. An example of the molecular species 34:1 is a molecule of formula (1) in which R1 has 16 carbon atoms, R2 has 16 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 1. The molecular species 34:2 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 32, and the total number of unsaturated bonds in R1 and R2 is 2. An example of the molecular species 34:2 is a molecule of formula (1) in which R1 has 16 carbon atoms, R2 has 16 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 2. The molecular species 34:3 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 32, and the total number of unsaturated bonds in R1 and R2 is 3. An example of the molecular species 34:3 is a molecule of formula (1) in which R1 has 16 carbon atoms, R2 has 16 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 3.

[0020] The molecular species 36:1 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 34, and the total number of unsaturated bonds in R1 and R2 is 1. An example of the molecular species 36:1 is a molecule of formula (1) in which R1 has 17 carbon atoms, R2 has 17 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 1. The molecular species 36:2 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 34, and the total number of unsaturated bonds in R1 and R2 is 2. An example of the molecular species 36:2 is a molecule of formula (1) in which R1 has 17 carbon atoms, R2 has 17 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 2. The molecular species 36:3 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 34, and the total number of unsaturated bonds in R1 and R2 is 3. An example of the molecular species 36:3 is a molecule of formula (1) in which R1 has 17 carbon atoms, R2 has 17 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 3. The molecular species 36:4 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 34, and the total number of unsaturated bonds in R1 and R2 is 4. An example of the molecular species 36:4 is a molecule of formula (1) in which R1 has 17 carbon atoms, R2 has 17 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 4. The molecular species 36:5 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 34, and the total number of unsaturated bonds in R1 and R2 is 5. An example of the molecular species 36:5 is a molecule of formula (1) in which R1 has 17 carbon atoms, R2 has 17 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 5. The molecular species 36:6 ​​of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 34, and the total number of unsaturated bonds in R1 and R2 is 6. An example of the molecular species 36:6 ​​is a molecule of formula (1) in which R1 has 17 carbon atoms, R2 has 17 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 6.

[0021] The molecular species 38:4 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 36, and the total number of unsaturated bonds in R1 and R2 is 4. An example of the molecular species 38:4 is a molecule of formula (1) in which R1 has 18 carbon atoms, R2 has 18 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 4. The phosphatidylcholine molecular species 38:5 indicates that the total number of carbon atoms in R1 and R2 is 36, and the total number of unsaturated bonds in R1 and R2 is 5. An example of the molecular species 38:5 is a molecule of formula (1) in which R1 has 18 carbon atoms, R2 has 18 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 5. The molecular species 38:6 of phosphatidylcholine means that the total number of carbon atoms in R1 and R2 is 36, and the total number of unsaturated bonds in R1 and R2 is 6. An example of the molecular species 38:6 is a molecule of formula (1) in which R1 has 18 carbon atoms, R2 has 18 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 6.

[0022] The phosphatidylcholine molecular species 40:6 indicates that the total number of carbon atoms in R1 and R2 is 38, and the total number of unsaturated bonds in R1 and R2 is 6. An example of the molecular species 40:6 is a molecule of formula (1) in which R1 has 19 carbon atoms, R2 has 19 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 6. The phosphatidylcholine molecular species 40:7 indicates that the total number of carbon atoms in R1 and R2 is 38, and the total number of unsaturated bonds in R1 and R2 is 7. An example of the molecular species 40:7 is a molecule of formula (1) in which R1 has 19 carbon atoms, R2 has 19 carbon atoms, and the total number of unsaturated bonds in R1 and R2 is 7.

[0023] From June 2023, the conventional non-alcoholic steatohepatitis (NASH) will be diagnosed as metabolic dysfunction associated steatohepatitis (MASH). In this specification, non-alcoholic steatohepatitis (NASH) can be read as metabolic dysfunction associated steatohepatitis (MASH).

[0024] The present invention relates to a method for assisting in the diagnosis of non-alcoholic steatohepatitis, comprising step 1 of measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample, and step 2 of calculating the ratio between the amount of LDL and the amount of phosphatidylcholine in the LDL.The present invention further relates to a method for obtaining data for assisting in the diagnosis of non-alcoholic steatohepatitis, comprising step 1 of measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample, and step 2 of calculating the ratio between the amount of LDL and the amount of phosphatidylcholine in the LDL.In this specification, the "method for assisting in the diagnosis of non-alcoholic steatohepatitis" and the "method for obtaining data for assisting in the diagnosis of non-alcoholic steatohepatitis" are collectively referred to as the methods of the present invention.

[0025] In the present invention, the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample are measured, and the diagnosis of NASH is supported based on the ratio of the amount of LDL to the amount of phosphatidylcholine in the LDL.The above-mentioned prior art documents do not describe measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL, and a method for supporting the diagnosis of NASH using the ratio of the amount of LDL to the amount of phosphatidylcholine in the LDL as an index has not been known until now.

[0026] The method of the present invention uses a specimen such as a blood sample, which is less invasive than liver biopsy and allows for a comprehensive understanding of the pathology of the entire liver without relying on a specific sample excised from a liver. Furthermore, specimens such as blood samples are easily collected, and if an automated analyzer-compatible measurement kit is used to measure the amount of LDL in the specimen and the amount of phosphatidylcholine in the LDL, measurements can be performed with simple procedures comparable to those used in daily medical care or health checkups.

[0027] <Specimen> The specimen may be any specimen derived from a subject animal, such as a blood sample (e.g., whole blood, serum, or plasma), urine, saliva, cerebrospinal fluid, tissue fluid, sweat, tears, amniotic fluid, bone marrow fluid, pleural effusion, ascites, indirect fluid, aqueous humor, and vitreous humor. Blood samples (e.g., whole blood, serum, or plasma) are preferred, with serum being particularly preferred.

[0028] The subject animals include mammals such as humans, monkeys, mice, rats, dogs, cats, pigs, and rabbits, with humans being more preferred.

[0029] The method for obtaining (collecting) a specimen from a test animal is not particularly limited, and for example, a specimen can be obtained (collected) from a test animal based on a method known per se, and may be subjected to separation, concentration, purification, etc. according to a method known per se, as necessary. Furthermore, the specimen may be one immediately after collection from the test animal, or may be a preserved specimen. The method for preserving the specimen may be any method commonly used in this field.

[0030] <Process 1> Step 1 is a step of measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample.

[0031] Methods for measuring the amount of LDL in a sample include a method for measuring the amount of LDL cholesterol, a method for measuring the amount of protein in LDL, and a method for measuring ApoB protein in a sample. A conventionally known method can be used to measure the amount of phosphatidylcholine in LDL in the sample. For example, a method can be used in which LDL is separated by a fractionation procedure such as high-performance liquid chromatography, ultracentrifugation, or electrophoresis, and then LDL and phosphatidylcholine are quantified by a quantitative procedure.

[0032] In this specification, the amount of LDL cholesterol means the amount of LDL-C, which is the amount of cholesterol (C) present in low-density lipoprotein (LDL).

[0033] The amount of LDL cholesterol (i.e., the amount of LDL-C) can be measured using the methods described in WO96 / 28734, JP-A-10-038888, JP-A-9-313200, WO07 / 007443, etc., but methods other than the above may also be used. The amount of LDL cholesterol and the amount of phosphatidylcholine in LDL can be measured using any method commonly used in this field, specifically, a method using a mass spectrometer or a method using an immunological assay, etc.

[0034] The method for measuring the amount of protein in LDL can be any method commonly used in this field. Specifically, LDL can be fractionated by ultracentrifugation, electrophoresis, column chromatography, etc., and the amount of protein in the fractions can be measured by the Lowry method, etc. It is preferable to fractionate LDL by ultracentrifugation, and then measure the amount of protein in the fractions by the Lowry method.

[0035] The method for measuring ApoB protein in a sample may be any method commonly used in this field, and specific examples include ELISA, immunoturbidimetry, and HPLC.

[0036] When measuring the amount of LDL and the amount of phosphatidylcholine in LDL using a method that utilizes a mass spectrometer, the method can be carried out by injecting a mobile phase (an organic solvent-based mobile phase such as acetonitrile, methanol, tetrahydrofuran, isopropanol, or ethanol, or an aqueous mobile phase such as water, phosphoric acid, formic acid, or acetic acid) and a sample into a separation and analysis device (e.g., a high-performance liquid chromatography device) equipped with a column [e.g., a gel filtration column], and separating and detecting LDL and phosphatidylcholine in LDL.

[0037] The specific method of using a mass spectrometer may be carried out according to a method known per se.

[0038] Specific examples of methods using a mass spectrometer include methods using high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography mass spectrometers (LC / MS), capillary electrophoresis mass spectrometers (CE / MS), gas chromatography mass spectrometers (GC / MS), inductively coupled plasma mass spectrometers (ICP / MS), liquid chromatography tandem mass spectrometers (LC / MS / MS), and gas chromatography tandem mass spectrometers (GC / MS / MS). Of these, methods using a liquid chromatography mass spectrometer (LC / MS) or a liquid chromatography tandem mass spectrometer (LC / MS / MS) are preferred, and methods using a liquid chromatography tandem mass spectrometer (LC / MS / MS) are more preferred.

[0039] When measuring the amount of LDL by immunological assay, a substance having affinity for LDL is used to form a complex between LDL and the substance having affinity for LDL, and then the complex is measured.

[0040] When measuring the amount of phosphatidylcholine in LDL by immunological assay, a substance having affinity for phosphatidylcholine is used to form a complex between phosphatidylcholine and the substance having affinity for phosphatidylcholine, and the complex is then measured.

[0041] The specific method of the immunological assay may be performed according to a method known per se.

[0042] Examples of immunological assays include enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), chemiluminescent enzyme immunoassay (CLEIA), electrochemiluminescent immunoassay (ECLEIA), immunoturbidimetry, immunonephelometry, latex agglutination, immunochromatography, Western blotting, Luminescent Oxygen Channeling Immunoassay (LOCI), and Liquid-phase Binding Assay-Electro Kinetic Analyte Transport Assay (LBA-EATA). Of these, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), chemiluminescent enzyme immunoassay (CLEIA), electrochemiluminescent immunoassay (ECLEIA), immunoturbidimetry, latex agglutination, and immunochromatography are preferred.

[0043] Specific examples of substances having affinity for LDL cholesterol and substances having affinity for phosphatidylcholine include antibodies. The antibody may be either a polyclonal antibody or a monoclonal antibody, and these may be used alone or in combination. The antibody may be an antibody fragment such as Fab, F(ab'2), Fv, or sFv, or a synthetic antibody such as a diabody, triabody, or tetrabody. The antibody to be used may be a commercially available antibody or one prepared according to a method known per se.

[0044] The anti-LDL antibody and the anti-phosphatidylcholine antibody may be labeled with a labeling substance.

[0045] Specific examples of labeling substances include enzymes such as horseradish peroxidase (HRP), bovine small intestinal alkaline phosphatase, and β-galactosidase; 99m Tc, 131 I, 125 I, 14 C.3 H, 32 P, 35 Examples of suitable materials include radioisotopes such as S, fluorescent substances such as fluorescein, fluorescein isothiocyanate (FITC), 4-methylumbelliferone, rhodamine, and derivatives thereof, luminescent substances such as luciferin, luminol, and ruthenium complexes, substances that absorb in the ultraviolet region such as phenol, naphthol, anthracene, and derivatives thereof, substances that have properties as spin labeling agents, typified by compounds having an oxyl group such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, dyes such as HiLyte dyes, Alexa dyes, and CyDye dyes, and nanoparticles such as gold colloids and quantum dots.

[0046] The labeling substance may be bound to the anti-LDL antibody and the anti-phosphatidylcholine antibody by a method known per se.

[0047] The amount of LDL and the amount of phosphatidylcholine in LDL may be expressed as either absolute values ​​such as volume or mass, or relative values ​​such as concentration, ionic strength, absorbance, fluorescence intensity, or turbidity.

[0048] <Process 2> Step 2 is a step of calculating the ratio between the amount of LDL and the amount of phosphatidylcholine in LDL. Specifically, the ratio between the amount of LDL and the amount of phosphatidylcholine in LDL can be calculated by dividing the amount of phosphatidylcholine in LDL obtained in step 1 by the amount of LDL obtained in step 1.

[0049] <Method to assist in the diagnosis of nonalcoholic steatohepatitis> In the method for assisting in the diagnosis of non-alcoholic steatohepatitis, the diagnosis of non-alcoholic steatohepatitis can be assisted based on the ratio of the amount of LDL calculated in step 2 to the amount of phosphatidylcholine in the LDL.

[0050] In the present invention, preferably, the ratio of the amount of LDL to the amount of phosphatidylcholine in the LDL is used as an index, and if the ratio is equal to or greater than the ratio of the amount of LDL in a sample from a patient suffering from simple fatty liver to the amount of phosphatidylcholine in the LDL in the sample, it can be determined that this indicates a high possibility of the patient developing non-alcoholic steatohepatitis.

[0051] In the present invention, it is preferable that when the ratio of the amount of LDL to the amount of phosphatidylcholine in LDL is equal to or greater than the cutoff value, it can be determined that this indicates a high possibility of developing non-alcoholic steatohepatitis.

[0052] That is, when the ratio of the amount of LDL to the amount of phosphatidylcholine in LDL is equal to or greater than a predetermined cutoff value, it can be determined that "the test animal is at risk of NASH, or the test animal is at high risk of NASH," etc. On the other hand, when the value derived from the test animal is less than the predetermined cutoff value, it can be determined that "the test animal is not at risk of NASH, or the test animal is at low risk of NASH," etc.

[0053] The cutoff value can be determined based on statistical analysis such as ROC (Receiver Operating Characteristic) curve analysis using the values ​​obtained in steps 1 and 2 using a sample derived from an animal suffering from NASH and the values ​​obtained in steps 1 and 2 using a sample derived from a healthy animal.

[0054] The sensitivity and / or specificity of the determination based on the cutoff value is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0055] According to the present invention, it is possible to preferably assist in distinguishing and determining non-alcoholic steatohepatitis from simple fatty liver.

[0056] The phosphatidylcholine in LDL is not particularly limited, and may be, for example, total phosphatidylcholine, or one or more types of phosphatidylcholine in which the total number of carbon atoms in the hydrocarbon groups constituting the fatty acids in the phosphatidylcholine is 30 to 40 and the total number of double bonds in the hydrocarbon groups constituting the fatty acids is 0 to 7.

[0057] The assisting method according to the present invention can be used as a method to assist doctors and others in diagnosing NASH.

[0058] <Specific examples of methods to assist in judgment> A specific example of the method for assisting the determination of the present invention will be described below. (1) Process 1 A sample (e.g., serum) is collected from a test animal (e.g., a human). The amount of LDL is measured. The amount of phosphatidylcholine (also known as LDL-PC) in LDL is measured by liquid chromatography tandem mass spectrometry (LC-MS / MS). In LC-MS / MS, the sample is injected into a separation analysis device (e.g., a high-performance liquid chromatography device) equipped with a gel filtration column, and the components in the sample are separated by using a mobile phase and flowing at an appropriate flow rate. The amount of phosphatidylcholine in the sample is then measured using a mass spectrometer for MS analysis.

[0059] (2) Process 2 The LDL-PC / LDL-C ratio is calculated by dividing the amount of LDL-PC measured in step 1 by the amount of LDL cholesterol.

[0060] (3) Judgment The value derived from the test animal calculated by (2) above is compared with a preset cutoff value, and if the value derived from the test animal is equal to or greater than the preset cutoff value, it is determined that "the test animal is at risk of NASH or has a high risk of NASH." On the other hand, if the value derived from the test animal is less than the preset cutoff value, it is determined that "the test animal is not at risk of NASH or has a low risk of NASH."

[0061] In a first embodiment, the amount of LDL cholesterol and the amount of LDL-PC in a sample are measured, and the LDL-PC / LDL-C ratio is calculated and used as an index. If the LDL-PC / LDL-C ratio in the sample is higher than the LDL-PC / LDL-C ratio in a sample from a patient with simple fatty liver disease (NAFL:SS), it can be determined that the patient is likely to have NASH (see Example 1). When the cutoff value for LDL-PC / LDL-C is set at 208.4 pmol / μg, the sensitivity is 77.8% and the specificity is 83.3%. The cutoff value used to determine whether a patient has NASH can be appropriately set within a range depending on the sensitivity and specificity desired by the user of the present invention. For example, a value between 200 and 220 pmol / μg can be used as the cutoff value.

[0062] In a second embodiment, the amounts of the LDL-PC molecular species 32:0, 32:2, and 34:3 per μg of protein in the LDL fraction in a sample are measured by liquid chromatography-mass spectrometry (LC-MS / MS). Using this as an indicator, if the amounts of the LDL-PC molecular species 32:0, 32:2, and 34:3 per μg of protein in the LDL fraction in a sample are equal to or greater than those in a sample from a patient with simple fatty liver disease (also known as NAFL:SS), it can be determined that the patient is likely to have NASH (see Example 2). When the cutoff value was set at 924.1 pmol / μg protein, the sensitivity was 88.9% and the specificity was 83.3%. The AUCs for the LDL-PC molecular species 32:0, 32:2, and 34:3 were 0.93, 0.87, and 0.87, respectively. When the cutoff values ​​were set at 14.3, 2.8, and 10.0 pmol / μg, respectively, the sensitivity was 77.8% and the specificity was 83.3%.

[0063] In the third embodiment, the amounts of the LDL-PC molecular species 30:0, 32:0, 32:1, 32:2, 34:3, 36:1, 36:2, 36:3, 36:4, 36:5, 36:6, 38:4, 38:5, 38:6, 40:6, and 40:7 in a sample are measured per μg of protein in the LDL fraction by liquid chromatography mass spectrometry (LC-MS / MS). When the total amount of double-bonded unsaturated fatty acyl chains (USFA) 32:1, 32:2, 34:3, 36:1, 36:2, 36:3, 36:4, 36:5, 36:6, 38:4, 38:5, 38:6, 40:6, and 40:7 (LDL-PC with USFA) per μg of protein in the LDL fraction is equal to or greater than the amount in samples from patients with simple fatty liver disease (NAFL:SS) (see Example 3), it can be determined that a patient has a high likelihood of developing NASH. Alternatively, when the total amount of 30:0 and 32:0 (LDL-PC without USFA) per μg of protein in the LDL fraction is equal to or greater than the amount in samples from patients with simple fatty liver disease (NAFL:SS), it can be determined that a patient has a high likelihood of developing NASH (see Example 3). The AUC for LDL-PC with USFA was 0.83, and the sensitivity and specificity were 88.9% and 83.3% at a cutoff value of 908.9 pmol / μg protein, respectively. The AUC for LDL-PC without USFA was 0.96, and the sensitivity and specificity were 88.9% and 83.3% at a cutoff value of 14.3 pmol / μg protein, respectively.

[0064] In a fourth embodiment, the total amount of polyunsaturated fatty acyl chain (PUFA) molecular species with multiple double bonds (LDL-PC 34:2, 34:3, 36:2, 36:3, 36:4, 36:5, 36:6, 38:4, 38:5, 38:6, 40:6, 40:7) (LDL-PC with PUFA) and the remaining LDL-PC without PUFA (LDL-PC 30:0, 32:0, 32:1, 34:1, 36:1) are measured by liquid chromatography mass spectrometry (LC-MS / MS) to determine the amount of the above molecular species of LDL-PC per μg of protein in the LDL fraction. For the total amount of polyunsaturated fatty acid chains (PUFA) molecular species with multiple double bonds (LDL-PC 34:2, 34:3, 36:2, 36:3, 36:4, 36:5, 36:6, 38:4, 38:5, 38:6, 40:6, 40:7) (LDL-PC with PUFA), if the amount per μg of protein in the LDL fraction is equal to or greater than the amount in samples from patients with simple fatty liver disease (NAFL:SS), it can be determined that a patient has a high likelihood of developing NASH (see Example 4).Alternatively, for LDL-PC without PUFA (LDL-PC 30:0, 32:0, 32:1, 34:1, 36:1), if the amount per μg of protein in the LDL fraction is equal to or greater than the amount in samples from patients with simple fatty liver disease (NAFL:SS), it can be determined that a patient has a high likelihood of developing NASH (see Example 4). The AUCs for LDL-PC with PUFA and LDL-PC without PUFA were 1.00 and 0.83, respectively. When the cutoff values ​​were set at 614.7 and 262.8 pmol / μg protein, respectively, the sensitivity was 100.0 and 66.7%, and the specificity was 80.0 and 83.3%, respectively.

[0065] <Methods for obtaining data to assist in the diagnosis of nonalcoholic steatohepatitis> The method of the present invention for obtaining data to assist in the diagnosis of non-alcoholic steatohepatitis (hereinafter also referred to as the method of the present invention for obtaining data) includes step 1 of measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample, and step 2 of calculating the ratio between the amount of LDL and the amount of phosphatidylcholine in the LDL.

[0066] Examples of data in the method for obtaining data of the present invention include (i) values ​​calculated in step 2 in the method for obtaining data of the present invention, (ii) values ​​obtained by further subjecting the above values ​​to multivariate analysis such as multiple logistic regression analysis, discriminant analysis, Poisson regression analysis, multiple regression analysis, Cox's proportional hazards model, path analysis, etc., (iii) data (comparison results) showing the magnitude relationship between the value calculated in step 2 of the method for obtaining data of the present invention and the above-mentioned cutoff value, and (iv) data suggesting that the subject animal is at risk of non-alcoholic steatohepatitis or that the subject animal is at high risk of non-alcoholic steatohepatitis, etc., with (i) or (iii) being preferred, and (i) being more preferred.

[0067] Steps 1 and 2 in the method for obtaining data of the present invention are as described above in this specification, and preferred examples, specific examples, etc. are also the same.

[0068] <Marker for determining non-alcoholic steatohepatitis> The ratio of the amount of LDL in a sample to the amount of phosphatidylcholine in the LDL in the sample is useful as a marker for determining non-alcoholic steatohepatitis. That is, according to the present invention, the ratio of the amount of LDL in a sample to the amount of phosphatidylcholine in the LDL in the sample is provided as a marker for determining non-alcoholic steatohepatitis. Furthermore, according to the present invention, the use of the ratio of the amount of LDL in a sample to the amount of phosphatidylcholine in the LDL in the sample is provided as a marker for determining non-alcoholic steatohepatitis.

[0069] <Methods for diagnosing and treating nonalcoholic steatohepatitis> According to another aspect of the present invention, there is provided a method for diagnosing and treating non-alcoholic steatohepatitis (hereinafter also referred to as the treatment method of the present invention).

[0070] The treatment method of the present invention comprises: Step 1: measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample; Step 2: calculating the ratio of the amount of LDL to the amount of phosphatidylcholine in LDL; Step 3 of determining non-alcoholic steatohepatitis based on the calculation result of step 2; and The method includes step 4 of administering appropriate treatment to patients who have been determined to be at risk of non-alcoholic steatohepatitis or to be at high risk of non-alcoholic steatohepatitis based on the determination result of step 3 above.

[0071] Steps 1, 2 and 3 in the treatment method of the present invention are as explained in the present specification, and preferred examples, specific examples and the like are also the same.

[0072] Suitable treatments in step 4 of the treatment method of the present invention include dietary therapy, exercise therapy, drug therapy, iron removal therapy, and surgical therapy. Exercise therapy includes aerobic exercise and resistance exercise. Drug therapy, depending on the pathological condition, includes the administration of one or more of the following drugs: (1) antioxidants (vitamin E, etc.), (2) antidiabetic drugs (thiazolidinediones, biguanides, citagliptin, etc.), (3) antihyperlipidemic drugs (fibrates, ezetimibe, EPL, etc.), or (4) liver protectors (urso, glycyrrhizin, etc.). Iron removal therapy includes phlebotomy or an iron-restricted diet. Surgical treatment includes weight loss surgery or liver transplantation.

[0073] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0074] Example 1: Six serum samples from patients with simple fatty liver (SS) and nine from patients with NASH (NASH) were analyzed for LDL-C and LDL-PC levels per 1dL of serum, and the LDL-PC / LDL-C ratio was calculated and compared between groups. LDL-C levels were measured using the Seiken LDL-EX(N) (Denka Seiken). LDL-PC levels were measured by liquid chromatography-mass spectrometry (LC-MS / MS). Patients were diagnosed with liver biopsy. Serum samples were separated by ultracentrifugation (50,000 rpm, 20 h, 4°C; Optima MAX Ultracentrifuge, Beckman Coulter) using a near-vertical rotor (MLN-80, Beckman Coulter) and gel filtration chromatography (Prominence Liquid Chromatograph, Shimadzu) using two Superose 6 columns (GE Healthcare). The eluent was 50 mmol / L phosphate-buffered saline (pH 7.4), and the injection volume was 0.3 mL. The flow rate was set at 0.5 mL / min, and 0.5 mL of each eluted fraction was collected. The LDL fraction was collected between 20 and 27 minutes (10.0–13.5 mL of eluate). 0.375 mL of each fraction was pooled to obtain the LDL solution, and its protein concentration was quantified by the Lowry method. Lipids were extracted from each LDL for simultaneous lipid analysis by liquid chromatography-mass spectrometry (LC-MS / MS) using an LTQ Orbitrap XL (Thermo Fisher Scientific). Each LDL fraction was adjusted to 2 mL with water. Next, 100 μL of a solution prepared by diluting SPLASH LIPIDPMIX Mass Spec Standard (Avanti) stock solution 50-fold with methanol as an internal standard (IS), 300 μL of methanol, and 2 mL of chloroform were mixed using a vortex mixer and then centrifuged at 3,500 rpm for 10 minutes at 4° C. The chloroform layer was collected, and 2 mL of chloroform was added to the remaining layer for extraction.The extract was evaporated to dryness, mixed with 300 μL of methanol, and centrifuged at 15,000 rpm for 10 minutes at 4°C. The supernatant was used as the LC-MS / MS sample. Lipids extracted from each LDL were measured using an Orbitrap LC-MS / MS system. Liquid chromatography (Shimadzu Corporation) was performed using an Atlantis T3 column (Waters). Elution was performed using a gradient of 5 mmol / L aqueous ammonium acetate, isopropanol, and methanol as the mobile phase. The gradient conditions are shown in Table 1. Measurements were performed using electrospray ionization in negative ion mode. Peak areas of the target lipid species and those used in the IS were calculated using Xcalibur 2.2 (Thermo Fisher Scientific). In Example 1, the peak areas were corrected for the IS peak area and the amount of serum used for LDL separation. In Examples 2, 3, and 4, the peak areas were corrected for the IS peak area and the amount of each protein in the LDL solution. All statistical analyses were performed using GraphPad Prism V7.0 / 10.1.2 software, and the area under the receiver operating characteristic curve (ROC-AUC) was calculated. The Mann-Whitney U test was used to compare molecular species levels between groups. The comparison graph is shown in Figure 1. The results showed that the LDL-PC / LDL-C ratio in the NASH group was significantly higher than that in the simple fatty liver group ( ). * P<0.05). Figure 2 shows the results of ROC analysis. The AUC was 0.87, which was favorable. When the cutoff value for LDL-PC / LDL-C in this population was set at 208.4 pmol / μg, the sensitivity was 77.8% and the specificity was 83.3%. [Table 1]

[0075] Example 2: The amounts of total LDL-PC and LDL-PC molecular species 32:0, 32:2, and 34:3 were measured using the 15 samples described in Example 1. Total LDL-PC and LDL-PC molecular species 32:0, 32:2, and 34:3 per μg of LDL fraction protein were analyzed using the liquid chromatography mass spectrometry (LC-MS / MS) data from Example 1. The comparison graphs are shown in Figures 3-6. As a result, LDL-PC, LDL-PC32:0, 32:2, and 34:3 in the NASH group were significantly higher than in the simple fatty liver group (denoted SS in the graph). * P<0.05, ** P<0.01). The results of ROC analysis are shown in Figures 7-10. The AUC for total LDL-PC was favorable at 0.83, and when the cutoff value was set at 924.1 pmol / μg protein, the sensitivity was 88.9% and the specificity was 83.3%. The AUCs for the LDL-PC molecular species 32:0, 32:2, and 34:3 were favorable at 0.93, 0.87, and 0.87, respectively. Furthermore, when the cutoff values ​​were set at 14.3, 2.8, and 10.0 pmol / μg, the sensitivity was 77.8% and the specificity was 83.3%.

[0076] Example 3: Using the 15 samples described in Example 1, in addition to the molecular species described in Example 2, the LDL fraction was analyzed for 30:0, 32:0, 32:1, 32:2, 34:1, 34:2, 34:3, 36:1, 36:2, 36:3, 36:4, 36:5, 36:6, 38:4, 38:5, 38:6, 40:6, and 40:7 per μg of protein using the liquid chromatography mass spectrometry (LC-MS / MS) data described in Example 1. Figures 11-12 show the results of a comparison between the simple fatty liver group (SS in the graph) and NASH for the total amount of unsaturated fatty acyl chains (USFA) with double bonds 32:1, 32:2, 34:1, 34:2, 34:3, 36:1, 36:2, 36:3, 36:4, 36:5, 36:6, 38:4, 38:5, 38:6, 40:6, and 40:7 (LDL-PC with USFA) and the total amount of the other 30:0 and 32:0 (LDL-PC without USFA). The results showed that the LDL-PC with USFA and LDL-PC without USFA in the NASH group were significantly higher than those in the simple fatty liver group ( * P<0.05, ** P<0.01). Furthermore, the results of ROC analysis are shown in Figures 13-14. The AUC for LDL-PC with USFA was favorable at 0.83, and when the cutoff value was set at 908.9 pmol / μg protein, the sensitivity was 88.9% and the specificity was 83.3%. The AUC for LDL-PC without USFA was favorable at 0.96. Furthermore, when the cutoff value was set at 14.3 pmol / μg, the sensitivity was 88.9% and the specificity was 83.3%.

[0077] Example 4: Using the 15 samples described in Example 1, the molecular species measured in Example 3 were divided into the total amount of polyunsaturated fatty acyl chains (PUFA) molecular species with multiple double bonds (LDL-PC 34:2, 34:3, 36:2, 36:3, 36:4, 36:5, 36:6, 38:4, 38:5, 38:6, 40:6, 40:7) (LDL-PC with PUFA) and the other LDL-PC without PUFA (LDL-PC 30:0, 32:0, 32:1, 34:1, 36:1), and the graphs comparing the NASH group and the simple fatty liver group (denoted SS in the graph) are shown in Figures 15-16. As a result, both the LDL-PC with PUFA and LDL-PC without PUFA in the NASH group were significantly higher than in the simple fatty liver group ( * P<0.05, ** P<0.01). The results of further ROC analysis are shown in Figures 17-18. The AUCs for LDL-PC with PUFA and LDL-PC without PUFA were favorable, at 1.00 and 0.83, respectively. When the cutoff values ​​were set at 614.7 and 262.8 pmol / μg protein, respectively, the sensitivity was 100.0% and 66.7%, and the specificity was 80.0% and 83.3%, respectively.

Claims

1. Step 1: Measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample; Step 2: Calculating the ratio of the amount of LDL to the amount of phosphatidylcholine in LDL A method for assisting in the determination of non-alcoholic steatohepatitis, comprising:

2. The method according to claim 1, wherein a ratio of the amount of LDL to the amount of phosphatidylcholine in LDL equal to or greater than a cutoff value indicates a high possibility of developing nonalcoholic steatohepatitis.

3. 2. The method of claim 1, wherein the ratio of the amount of LDL to the amount of phosphatidylcholine in the LDL is used as an index, and if said ratio is equal to or greater than the ratio of the amount of LDL in a sample from a patient suffering from simple fatty liver to the amount of phosphatidylcholine in the LDL in said sample, it indicates a high possibility that the patient has developed non-alcoholic steatohepatitis.

4. Phosphatidylcholine in LDL total phosphatidylcholine, or one or more types of phosphatidylcholine in which the total number of carbon atoms in the hydrocarbon groups constituting the fatty acids in the phosphatidylcholine is 30 to 40 and the total number of double bonds in the hydrocarbon groups constituting the fatty acids is 0 to 7; The method of claim 1.

5. 2. The method of claim 1, which aids in distinguishing nonalcoholic steatohepatitis from simple fatty liver.

6. The method of claim 1 , wherein the specimen is a blood sample.

7. The method of claim 1 , wherein the sample is whole blood, serum, or plasma.

8. Step 1: Measuring the amount of LDL in a sample and the amount of phosphatidylcholine in the LDL in the sample; Step 2: Calculating the ratio of the amount of LDL to the amount of phosphatidylcholine in LDL A method for obtaining data to assist in the diagnosis of non-alcoholic steatohepatitis, comprising:

9. Phosphatidylcholine in LDL total phosphatidylcholine, or one or more kinds of phosphatidylcholines in which the total number of carbon atoms in the hydrocarbon groups constituting the fatty acids in the phosphatidylcholines is 30 to 40 and the total number of double bonds in the hydrogen groups constituting the fatty acids is 0 to 7; The method of claim 8.

10. The method of claim 8 , wherein the specimen is a blood sample.

11. The method of claim 8, wherein the sample is whole blood, serum, or plasma.

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